The present invention is drawn to the genetic modification of higher plants using chimeric RNA/DNA oligonucleotides.
Because of the complex organization and metabolic compartmentation in higher plants, many molecular tools have to be combined to successfully genetically manipulate a plant. At the present time, directed changes require a transformation system, a suitable gene, a promoter sequence capable of driving expression of the gene, effective targeting signals to direct the expression product to the correct destination in the cell, and often other regulatory elements.
There are many published systems for the transformation of plant cells. However, there remain drawbacks to each of the systems. For example, Agrobacterium tumefaciensgene transfer is widely used for creating transgenic plants, however, in some plants making a successful transformation utilizing the Agrobacterium system is difficult. Other systems include particle bombardment, viral vectors, protoplast transformation via polyethylene glycol or electroporation, microinjection of DNA into protoplast, and macroinjection of DNA. Of these, the most successful method to date is particle bombardment of embryogenic calli for developing embryos. Even this transformation system suffers from a lack of genomic targeting control for insertion of the foreign gene. In addition, bombardment results in multiple insertions of the foreign gene into the plant genome.
Any transformation system in plants, must deal with the problems of cosuppression which occurs when endogenous genes are down-regulated by expression of homologous sense transcripts. Expression of chimeric gene constructs have led to the down-regulation of genes in transgenic plants, however the effects of cosuppression are variable, and the underlying mechanisms remain unclear.
Site-directed manipulation of chromosomal genes has become the method of choice for determining gene function and bacterial, yeast, and mammalian cells. The primary methods used in site-directed gene manipulation rely on gene replacement via homologous recombination using an appropriately designed gene targeting vector. In plants, gene targeting has been limited by the frequency of homologous recombination. Even with improvements in transformation and selection methods, the frequency of gene targeting in plants is still 10xe2x88x923-10xe2x88x924 fold lower than random integration.
New procedures are being developed for mammalian gene therapy. One example is an approach using chimeric RNA/DNA oligonucleotides. In mammalian cells, chimeric oligonucleotides that contain both DNA/DNA and RNA/DNA duplex regions with homology to a target locus, are capable of specifically correcting mutations at a high frequency in both episomal and chromosomal target genes. Recently, mutations in liver alkaline phosphatase gene and factor IX gene have also been efficiently corrected in human hepatoma cells. However, to date, chimeric oligonucleotide-based gene targeting has not been reported in plant systems.
Herbicide resistant forms of plants are desirable for many breeding and crop production applications. Approaches to date have involved laborious methods including: finding a naturally existing form of resistance in a plant and introgressing the trait into desirable germplasm; mutagenesis of plants, seeds, and seedlings to generate novel mutant plants that confer resistance and introgressing the trait into the breeding population; finding a naturally existing form of a gene which confers resistance to a target herbicide and introducing the gene into the desired species by transformation; and, converting a wild type gene to a resistant form by mutagenesis. All of these approaches rely on either natural recovery of the trait or modification of the gene and subsequent introduction of the resistance gene into a plant.
A major disadvantage in each of these approaches is the time involved in terms of mutagenesis, recovery of the trait and the breeding necessary to introduce the trait into desired populations. Further, where transformation is involved, plants will have to be tested and selected that are not impacted by expression instability or by poor agronomic performance. Additionally, in many instances, optimum performance of a gene in a given species may only be achieved following resynthesis of the gene to maximize usage of preferred codons, or by the creation of modified forms of the gene.
Because of the present problems associated with the integration and expression of foreign genes in plant cells, effective strategies for modification, conversion or correction of gene sequences are needed.
Compositions and methods for modifying nucleotide sequences of interest in a plant cell are provided. The compositions comprise single covalently linked duplex oligonucleotides, a portion of which are homologous to a plant nucleotide sequence of interest. The oligonucleotides comprise both deoxyribonucleotides and ribonucleotides. To create a modified plant sequence the oligonucleotides contain within the region of homology to the native plant sequence at least one noncorresponding or heterologous base pair that replaces the naturally occurring sequence in the plant genome.
The chimeric oligonucleotide used for target site conversion consists of a single-stranded nucleic acid designed to form a duplex structure capped by single-stranded loops. One strand of the duplex pairs with the target sequence of the plant. This strand consists of a deoxyribonucleotide stretch of nucleotides with at least one mismatch to the target sequence, flanked by two sequence-specific targeting segments of 2xe2x80x2-O-methyl ribonucleotides. The lower strand of the duplex contains only deoxyribonucleotides. Homologous pairing between the chimeric oligonucleotide and the plant""s target sequence and the strand transfer process are enhanced by the modified RNA residues.
The method involves converting or modifying the nucleotide sequence of interest in the plant cell by recombination or repair between homologous sequences, in the duplex oligonucleotide and the plant nucleotide sequence. The process exchanges the heterologous base pairs for the naturally occurring base pairs. Plants having the converted or modified sequence can be obtained. The methods are useful for making a variety of sequence-specific changes for applications such as site-specific mutagenesis, gene knockout, allelic replacement, and the like.
In a particular embodiment, compositions and methods for generating herbicide resistant plants are provided. The compositions comprise single covalently linked duplex oligonucleotides that are homologous with a nucleotide sequence of interest except for base pairs necessary to convert the sequence to a herbicide resistant form of the gene. The method involves converting the naturally occurring nucleotide sequence in a plant by targeted gene conversion to create a herbicide resistance form of the gene. Herbicide resistant plants can be obtained from the method.